CORE--GENOTYPING LABORATORY

University Of California San Francisco, San Francisco CA

Investigators

Linked publications & trials

Abstract

Accurate genotyping of the animals tested in other Projects is a vital part of this Program. The Genotyping Core will supply those services in an efficient, accurate and cost-effective manner. Because various genetic manipulations will provide the animals to be tested in the proposed Program, no single technique or strategy will cover all eventualities. Accordingly, a combination of several methodologies will be used to verify the transgenic or gen targeting events under consideration. These will include Southern blotting, restriction fragment length polymorphisms (RFLPs), polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) and double-stranded DNA sequencing. In addition, animals will be karyotyped if there are specific concerns about chromosomal abnormalities, such as extensive deletions. DNA will be extracted from each punches or tail clips provide by the other Projects. In traditional 'transgenic' animals, we will combine Southern blotting and RFLP approaches, utilizing restriction sites specifically engineered into the transgenic constructs In addition, specific primer pais may be employed to detect the presence of the transgene by PCR amplification of a selected region of the transgene of interest. In the case of the global knockout of a targeted locus, a marker gene is frequently incorporated into the genome as part of the targeting event, and PCR and RFLP can detect the presence of the marker gene. In the case of a 'conditional/ knockout of a targeted locus, a transgene such as cre can be detected by PCR, while the 'floxed' targeted gene is easily detected by screening for the gene targeting event by Souther blot/RFLT analysis or by PCR. For knock-in animals, it is crucial to detect the presence of the mutational in the coding region of the targeted gene. This will be done using a PCR, RFLP or SSCP strategy, or by direct sequencing.